The quantification of nucleic acids represents an important tool in many molecular biology applications, such as gene expression analyses. A common approach to the study of gene expression is the production of complementary DNA (cDNA), the technique of which employs the reverse transcription of RNA into the complementary DNA using the enzyme reverse transcriptase (RT): In order to have the RNA transcribed, RNA molecules from an organism are isolated from an extract of the cells or tissues of the organism. By using the enzyme reverse transcriptase (RT) or a DNA polymerases having RT activity, cDNA copies may be created from the RNA template, which results in the production of single-stranded cDNA molecules. In the reverse transcription reaction primers, dNTPs, and a suitable buffer is commonly needed. The primers anneal to the RNA template and are extended on their 3′-end complementary to the RNA template. In order to prevent RNA from degradation, commonly RNase-inhibitors are employed increasing the reliability and reproducibility of the cDNA synthesis.
The discovery of the described reverse transcription reaction has enabled development of sensitive molecular biological methods not only for basic research, but also for the design of medicaments and diagnostics.
Known reverse transcriptases are the Avian myoblastosis virus (AMV) reverse transcriptase, which was the first widely used RNA dependent DNA polymerase. The enzyme has 5′-3′ RNA dependent DNA polymerase activity, 5′-3′ DNA dependent DNA polymerase activity, and RNase H activity. RNase H is a processive 5′ and 3′ ribonuclease specific for the RNA strand for RNA/DNA hybrids. Also, reverse transcriptase originating from Moloney murine leukemia virus (M-MLV) and from human immunodeficiency virus type 1 (HIV-1) are used extensively in molecular biology.
In general, reverse transcriptase is a multifunctional enzyme with at least three enzymatic functional activities: (i) RNA-dependent DNA-Polymerase, (ii) DNA-dependent DNA-Polymerase, and (iii) RNA-DNA-hybrid-dependent RNAse (RNase H).
The reverse transcription reaction primarily utilizes the RNA-dependent polymerase activity for the generation of cDNA. This activity permits the in-vitro synthesis of cDNA for cloning and reverse transcriptase polymerase chain reactions, RNA-sequencing and primer extension experiments. Also, with this activity, an RNA template can only transcribed into one molecule of cDNA, and there is no amplification during the reverse transcription of the RNA sequence.
The RNase H activity specifically recognizes and degrades RNA:DNA hybrids. Thus, this activity does not affect pure RNA, but only RNA hybridized to the newly-synthesized cDNA. As a consequence, the RNA can be degraded by the RNase H only as early as the cDNA has been synthesized. Some of the reverse transcriptases presently available on the market have been mutated and have, thus, no significant RNase H activity. In case such RNase H deficient reverse transcriptases are being employed in reverse transcription reactions, and in case the cDNA generated in the reverse transcription reaction is, e.g, to be used for RT-PCR, a separate RNA-degradation step following reverse transcription reaction has to be performed by incubation with RNase H.
With the single-stranded cDNA as template, the DNA-dependent DNA polymerase activity synthesizes a complete double-stranded cDNA of the original mRNA. It is noted, however, that a premature synthesis of the second strand often leads to shortened double-stranded cDNAs, since—after uncompleted transcription—the reverse transcription reaction activity for the single-strand synthesis often switches into the second-strand synthesis activity. Thus, in the recent in-vitro reaction conditions the double-stranded cDNA-synthesis of the reverse transcriptase is usually suppressed.
The cDNA generated in the reverse transcriptase reaction can then be further characterized and quantified by methods such as cloning, sequencing, and polymerase chain reaction (PCR); the latter, i.e. PCR, exploits first-strand cDNA for mRNA sequence(s) as template for amplification by the PCR. This method is referred to as reverse transcriptase PCR (RT-PCR), which method is widely used for detection and quantification of RNA.
Ideally, the reverse transcription reaction synthesizing the cDNA—completely representing the applied RNA—should be unaffected by interfering factors whatsoever, and should, thus, represent a non-biased reaction. In reality, however, the efficiency of the reverse transcriptase reaction can vary greatly, depending on factors such as the intrinsic enzymatic properties of the reverse transcriptase, reaction buffer composition, reaction temperature and duration, and possible reverse transcriptase inhibitors present in the RNA sample to assess. All these factors may negatively affect the reverse transcriptase efficiency and may lead to shortened cDNA-molecules, consequently leading to a direct impact on the outcome of downstream analyses such as sequencing and PCR.
Although several different approaches have been made to improve overall efficiency of the reverse transcription conditions and, thus, of the cDNA synthesis as such, today, there still is the need for effectively optimizing the reverse transcriptase, buffers, temperature and other conditions, in particular when developing new reverse transcriptase products.
In view of the above, it is an object of the present invention to provide for tools by means of which reliable and reproducible information on reverse transcriptase kinetics and reverse transcription efficiency can be gained.
The present invention satisfies these and other needs.